American military veterans commonly develop unremitting pulmonary diseases in which the alveoli comprising the gas-exchange region are destroyed leading to frequent hospitalizations. Reductions in cigarette smoking have not been accompanied by reductions in health care costs and deaths related to these diseases, highlighting the need for treatments to allay further destruction and repair damaged alveoli. To develop such treatments it is important to identify factors which are active during development, and could be manipulated to repair alveoli in adults. The applicant has concentrated on interstitial lung fibroblasts (LF) during alveolar formation and now proposes investigations to elucidate how LF are directed to move away from their initial locus at eruptions from the primary septa into the airspace during secondary septation. Hypothesis: Platelet-derived growth factor receptor-alpha (PDGFRa) and sonic hedgehog (Shh) cooperatively regulate secondary alveolar septal elongation by fostering fibroblast polarization and directional migration. Specific Aim 1: To investigate how lung fibroblasts orient along the axis of the secondary septal elongation (polarize) and how polarization is influenced by PDGFRa and Shh. Specific Aim 2: To localize shared signaling molecules in the PDGF-A and Shh intracellular signaling pathways during LF migration in vitro, and define the topography of their activation in relationship to the primary cilium, centriole, and remodeling microtubules. Preliminary studies have shown that LF expressing PDGFRa preferentially localize towards the most distal portions of the alveolar septum, more abundantly display primary cilia, and orient their centrioles towards the alveolar entry ring (AER). PDGFRa regulates Akt, enhances proliferation, and reduces apoptosis in LF which express this receptor. Shh signals through a non-canonical pathway involving the G- protein coupled receptor properties of Smoothened. PDGF-A and Shh cooperatively enhance directional migration of LF and share important signaling intermediates that modify microtubules (MT) and direct cell- polarity. Studies are proposed to accomplish these objectives, including stereological analysis of lungs from mice in which one allele of the endogenous PDGFRa-promoter regulatory region drives expression of a green fluorescent protein (GFP) tag. This will enable localization and enumeration of this LF-population, and recognition of how the polarity of these cells differs from other alveolar cells, with respect to PDGF-A and Shh signaling. PDGF-A and Shh signaling will be perturbed using conditional deletions of PDGFRa or smoothened, or by inhibiting PDGFRa-kinase activity. Such perturbations will identify how these pathways ensure adequate and directed movement of LF, through regulation of their polarity during migration. Live cell, time-lapse imaging will be used to quantify the speed and persistence of LF migration in microfluidic devices in order to learn how these parameters are modified by PDGF-A, Shh and alterations in cell shape. Signaling events and MT-remodeling will be localized using immunofluorescence microscopy: both through immunohistochemistry and by tracking fluorescently labeled proteins expressed by a LF-cell line. Attention will be focused on pathways (phosphoinositol 3-kinase, PI3K and Akt/protein kinase-B) which maintain polarity by regulating microtubule stabilization at the leading edge. These proposed studies will illuminate how LF reach their optimal locations and persistently migrate along a properly oriented axis of alveolar septal elongation. Knowing how LF are directed to their optimal location may facilitate understanding how elastic fibers are deposited in a mechanically optimized pattern. Information from the proposed studies will also serve as a basis for future studies of how mechanical strain induced by respiration impacts LF positioning. Understanding basic structural and cellular pathways is required to identify and develop pharmacologic agents to promote alveolar repair in emphysema.